Characterization of Biomolecular assemblies
Native mass spectrometry is an information rich technique that is applied routinely in NovAliX to non-covalent protein/ligand complexes formed in solution. New and productive ways of confirming and characterizing hits and leads during the drug discovery process are thereby opened-up. The workflow for native MS in NovAliX is highly automated, consumes minimal amounts of sample and provides useful data: i) stoichiometry of the protein/ligand complex, ii) binding reversibility, iii) binding-site specificity, iv) complex affinity and v) gas-phase stability which relates to the extent of polar-based interactions involved between the protein and the ligand. These points are outlined in greater detail below :
- The binding stoichiometry is determined from the mass of bound versus free target. The difference in mass reveals the number of bound molecules.
- Binding reversibility is particularly important for drug discovery. This issue can be conveniently assessed using two ways: first by deliberately inducing the dissociation of non-covalent interactions in the mass spectrometer, secondly by diluting sample in a denaturing solvent to break-down the non-covalent frameworks in solution.
- Binding site specificity can be readily investigated by competition assays involving a reference ligand interacting in the targeted site. Complex displacement is uniquely evidenced from accurate mass measurements of the resulting protein/ligand complexes.
- Equilibrium dissociation constants (Kd) can be obtained by titration of the target protein with ligand of increasing concentration. The proportion of complex is determined from the relative peak heights in the mass spectra. Native MS experiments can be used to determine Kds extending from the high nanomolar up to the high double-digit micromolar range.
- A valuable feature of native MS is the possibility to reveal the presence of natural or fortuitously bound ligands occupying the target site. These would most likely interfere with screens or generate misleading data and trigger false conclusions.
- The nature of non-covalent interactions may be explored through gas phase dissociation experiments. Monitoring complex dissociation as a function of increasing acceleration voltage yields measurement of the protein/ligand complex stability in the gas-phase. Based on this empirical correlation, comparison of the stability of complexes in the gas phase provides a quick indication of possible gain or loss of polar interaction among a compound series during a hit-to-lead optimization campaign. For example, see [Vivat Hannah et al. Future Medicinal Chemistry 2010, (2), 35].